T cell receptor-MHC class I peptide interactions: affinity, kinetics, and specificity.

The critical discriminatory event in the activation of T lymphocytes bearing alpha beta T cell receptors (TCRs) is their interaction with a molecular complex consisting of a peptide bound to a major histocompatibility complex (MHC)-encoded class I or class II molecule on the surface of an antigen-presenting cell. The kinetics of binding were measured of a purified TCR to molecular complexes of a purified soluble analog of the murine MHC class I molecule H-2Ld (sH-2Ld) and a synthetic octamer peptide p2CL in a direct, real-time assay based on surface plasmon resonance. The kinetic dissociation rate of the MHC-peptide complex from the TCR was rapid (2.6 x 10(-2) second-1, corresponding to a half-time for dissociation of approximately 27 seconds), and the kinetic association rate was 2.1 x 10(5) M-1 second-1. The equilibrium constant for dissociation was approximately 10(-7) M. These values indicate that TCRs must interact with a multivalent array of MHC-peptide complexes to trigger T cell signaling.

Heterodimeric, disulfide-linked alpha/beta T cell receptors in solution.

Structural and functional analysis of T cell receptor (TcR)-ligand binding would be greatly advanced by the availability of an intact, assembled TcR in soluble form. We have produced such a molecule, by splicing the extracellular domains of a TcR to the glycosyl phosphatidylinositol membrane anchor sequences of Thy-1. The molecule is expressed in the absence of CD3 on the cell surface, and can be cleaved from the membrane by treatment with phosphatidylinositol-specific phospholipase C. The alpha and beta chains of the soluble molecule are paired in the native conformation as judged by reactivity with the anti-V beta 8 monoclonal antibody F23.1, and with the anti-clonotypic monoclonal antibody 1B2; it is a disulfide-linked dimer with a mol. mass of 95 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, and 47 kDa after reduction. We conclude that we have generated an alpha/beta TcR in soluble form.